Isolation of uteroevacuant substances from plant extracts

ABSTRACT

A method of obtaining uteroevacuant substances from the zoapatle plant is described. The isolation and purification of biologically-active compounds from the zoapatle plant is accomplished by chemical means.

In co-pending application Ser. No. 672,918, now U.S. Pat. No. 4,086,358a method is described for isolating and purifying the active principlesin the zoapatle plant. These materials have been shown to possessuteroevacuant properties. In the process described in the above-notedapplication, a crude extract is first obtained by a series of extractionand purification steps and the crude extract is further purified bychromatography over silicic acid. As a result of the chromatography, asemi-purified mixture containing at least three major components isobtained. This mixture is then chromatographed through a column of apolymeric gel. A number of fractions are collected and the compositionof each fraction is monitored by either gas chromatography or thin layerchromatography. As a result of the second chromatogram, two chemicallydistinct compounds are obtained as evidenced by gas chromatography andspectral analyses. These copounds have the following formulae. ##STR1##

The present invention relates to a chemical method of isolating,separating and purifying crude extracts or semi-purified mixturesobtained from the zoapatle plant to obtain the biologically-activecompounds.

The zoapatle plant is a bush about 2 m. high that grows wild in Mexico.Botanically it is known as Montanoa tomentosa according to Cervantes,Fam. Compositae, Tribe Heliantheae; another variety of the species isMontanoa floribunda. The plant is described in great detail in LasPlantas Medicinales de Mexico, third edition, Ediciones Botas (1944).

The plant has been used for centuries in the form of a "tea" or othercrude aqueous preparations primarily as a labor inducer or mensesinducer for humans. Its use as a uteroevacuant agent has been documentedin the literature, but definitive chemical and pharmacological studieshave not been described. By uteroevacuant is meant an agent which causesthe uterus of warm blooded animals to contract or expel its contents.Such agents are generally employed to induce menses, expel a hydatiformmole, expel or resorb a fetus, induce abortion or delayed labor and insituations in which the contents of the uterus, such as the fetus orplacenta, should be evacuated.

The process of the present invention is illustrated by the followingreaction sequence wherein R is an acyl group derived from an aliphaticacid. ##STR2##

As the starting material in the process of the present invention, eitherthe crude extract or the semi-purified material containing at leastthree major components obtained as described in Ser. No. 672,918 can beemployed. The semi-purified residue is first converted to the acylderivatives by reaction with an excess of an acyl halide or an acidanhydride. The acylation step is preferably carried out in the presenceof a base. Acyl halides or acid anhydrides having 1-10 carbon atoms inthe acyl group may be employed. The preferred acylating agents are theacetyl, propionyl, butyryl and valeryl halides and the correspondinganhyrides. Bases which may be employed in the acylation step includetrialkylamines such as triethylamine, trimethylamine and tripropylamine,and tertiary amines such as pyridine, N,N-dimethylaniline andN,N-diethylaniline. The reaction is preferably carried out in an inertatmosphere such as nitrogen or argon, for example, and at a temperaturebetween about 0°-30° C. The reaction may be carried out in an organicsolvent such as, for example, benzene, ether, tetrahydrofuran, tolueneand the like. The acylated materials are obtained from the reactionmixture by techniques known to those skilled in the art. For example,the organic base can be removed by extraction with a dilute aqueous acidsolution or an aqueous saturated copper sulfate solution. The crudemixture of acyl derivatives is then obtained by removal of the solvent.

Treatment of the mixture of acyl derivatives with an excess of reducingagent such as sodium borohydride or lithium tritertiarybutoxy aluminumhydride, for example, converts the compounds to the hydroxy derivatives(III-a and III-b). The reduction step is preferably carried out in aninert atmosphere, such as nitrogen or argon, and in an organic solventsuch as ethanol, methanol or tetrahydrofuran. The particular solventemployed will depend upon the particular reducing agent employed in thereaction. It is preferred to carry out the reaction at room temperature,however, temperatures as low as 0° C may be employed in the reductionstep. The reaction mixture is generally quenched with dilute aqueousacids such as, for example, dilute hydrochloric acid or a saturatedammonium chloride solution. After removal of the solvent, the crudemixture of hydroxy derivatives is used as such in the next step.

Treatment of the mixture of hydroxy derivatives with an oxidizing agentsuch as manganese dioxide selectively oxidizes III-b to II-b. It ispreferred to carry out the reaction at room temperature with an excessof the oxidizing agent, although elevated temperatures may be employed.The reaction may be carried out in an organic solvent such as methylenechloride, chloroform, hexane or benzene. The compounds are thenseparated by physical means. For example, the inorganic materials can beremoved by filtration and the residue left after removal of the solventcan be chromatographed over an adsorbent material such as silica gel toafford III-a and II-b.

The hydroxy derivative (III-a) is converted to the corresponding ketone(II-a) by treatment with a suitable oxidizing agent such as a mixture ofchromium trioxide-sulfuric acid and chromium trioxide-pyridine. Thereaction is preferably carried out in an inert atmosphere such asnitrogen or argon, for example, at a temperature of about 0° C, althoughlower temperatures may also be employed. The reaction is generallycarried out in an organic solvent such as acetone, 2-butanone, methylenechloride or chloroform, depending upon the particular oxidizing agentemployed. The acetyl derivative (II-a) is converted to the underivatizeduteroevacuant material (I-a) by hydrolysis with a suitable base such as,for example, sodium hydroxide, potassium hydroxide or tetra n-butylammonium hydroxide. The reaction is preferably carried out in an inertatmosphere such as nitrogen or argon at room temperature; however,temperatures as high as the reflux temperature of the solvent may alsobe employed. Methanol, ethanol, isopropanol, benzene, ether ortetrahydrofuran may be employed as the solvent. Aqueous media may alsobe employed. The crude product (I-a) can be further purified by columnchromatography over an adsorbent material such as silica gel, alumina orflorisil.

The acetyl derivative first obtained above (II-b) is converted to theunderivatized uteroevacuant material (I-b) by hydrolysis with a suitablebase as described above. The crude product (I-b) can be further purifiedby column chromatography over an adsorbent material such as silica gel,alumina or florisil. The presence of uteroevacuant materials in theproducts obtained from the reactions is determined through proceduresemployed for the detection of uterine contractions and interruption ofpregnancy. The compounds are identified by thin layer chromatography andspectral analysis.

The purified compounds are effective in inducing uterine contractionswhen administered in doses ranging from about 1.0 mg. to about 85mg./kg. The purified compounds are effective in interrupting pregnancyat dosage levels between about 15 to about 100 mg./kg. The preferreddosage range is from about 20-85 mg./kg. As central nervous systemdepressants, the compounds are active in doses as low as 3.7 mg./kg. Theactual dosage employed will depend upon the species of animal to whichthe compound is administered. The compounds can be administered informulations prepared according to acceptable pharmaceutical practices.Suitable formulations include solutions, suspensions and solid dosageforms in pharmaceutically acceptable carriers. They can be administeredperorally or intravenously or in any conventional manner in accordancewith acceptable pharmaceutical practices.

EXAMPLE I (A) Preparation of the Diacetate Derivatives (II-a and II-b)

The semi-purified starting material (1.172 g.) is dissolved in asolution of benzene (30 ml.) and pyridine (10 ml.) and the solution istreated while stirring at 0° C with acetyl chloride (5 ml.) undernitrogen. The reaction mixture is allowed to warm to room temperatureand is stirred for 4 hrs. The resulting mixture is treated with icewater (10 ml.) and ether (200 ml.). The organic layer is washed andsaturated cupric sulfate solution (3 × 100 ml.) and dried (MgSO₄). Afterremoval of the solvent in vacuo, the crude mixture of II-a and II-b(1.212 g.) is used as such in the next step.

(B) Sodium Borohydride Reduction

The mixture (1.212 g.) obtained in A above is dissolved in methanol (100ml.) and treated with sodium borohydride (400 mg.) at room temperatureunder nitrogen. The resulting mixture is stirred for 5 mins. and thentreated with a saturated ammonium chloride solution (100 ml.). Theaqueous mixture is extracted with methylene chloride (300 ml.). Theorganic layer is dried (MgSO₄) and then evaporated in vacuo to give ayellow oil (1.1 g.) The crude mixture of III-a and III-b is used as suchin the next step.

(C) Manganese Dioxide Oxidation

The crude mixture (III=a and III-b) obtained in B above (1.1 g.) isdissolved in methylene chloride (100 ml.) and the resulting solution istreated at room temperature with manganese dioxide (4 g.). The resultingmixture is stirred for 16 hrs. and then filtered through a pad ofcelite. The organic layer is dried (MgSO₄) and evaporated in vacuo toyield a yellow oil. The oil is chromatographed on a SilicAR (a neutralsilica gel product sold by Mallinckrodt, Inc., St. Louis, Missouri)column (20 g.). The keto derivative II-b (243 mg.) is eluted with 25:75ether-petroleum ether; the hydroxy derivative III-a (754 mg.) is elutedwith 75:25 ether-petroleum ether. The spectral characteristics of II-band III-a are as follows:

II-b - I.R. (Neat) μ : 5.75, 5.95, 6.2 and 8.1; N.M.R. (CDCl₃) δ: 6.03(bs, 1H, ##STR3## 5.35 (m, 1H, >C═CH--CH₂ --O); 4.62 (d, J=6Hz,>C═CH--CH₂ OAc); 4.08 (bs, 2H ##STR4## 2.03 (s, 6H, ##STR5##

III-a - I.R. (Neat) μ : 2.86 and 5.75;

N.m.r. (cdcl₃) δ: 5.3 (m, 2H, >C═CH--CH₂ OAc and ##STR6## 4.7 (d, J=6Hz,2H, >C═CH--CH₂ --OAc); 4.1 (bs, 2H ##STR7## 3.45 (m, 1H, Ac--O--CH<);2.03 (s, 6H, ##STR8##

(d) Jones Oxidation

The hydroxy derivative (III-a, 333 mg.) obtained as in C above isdissolved in acetone (5 ml.) and treated slowly with Jones reagent (2mmole) at 0° C under nitrogen. The resulting mixture is stirred for 7mins, and then treated with ether (30 ml.) and water (20 ml.). Thelayers are separated and the aqueous layer is extracted with ether (20ml.). The combined organic layer is washed with water (30 ml.), dried(MgSO₄) and evaporated in vacuo to give an oil. The crude product (II-a)is used as such as in the next step.

(E) Preparation of I-a

The crude product (II-a) obtained in D above (161 mg.) is dissolved intetrahydrofuran (5 ml.) and water (5 ml.). To this mixture, tetran-butyl ammonium hydroxide (20% solution in methanol, 1 ml.) is addedunder nitrogen at room temperature and the result mixture is stirred for40 hours. The mixture is treated with 50 ml. of ether and the organiclayer is washed with 10% hydrochloric acid (2 × 15 ml.), dried (MgSO₄)and evaporated in vacuo to give an oil. This crude product is purifiedby chromatography on a SilicAR column (5 g.). The compound I-a (81.8mg.) is eluted with ether. Its ir, nmr spectra, R_(f) on thin layer andretention time on gas chromatography are identical to those of compoundI reported in co-pending application Ser. No. 672,918. The compound hasthe following spectral characteristics:

I.R. (Neat) μ : 2.91 and 5.88; N.M.R. _(TMS) ^(CDCl).sbsp.3 δ : 5.41 (m,2H, >C═CH--CH₂ OH and ##STR9## 4.20 (d, 2H, >C═CH₁₃ CH₂ OH); 4.15 (s,2H, ##STR10## 3.58 [broad t, 1H, >CH(OH]; 3.18 (d, 2H, ##STR11## 1.71[d, 6H, >C═C--(CH₃)₂ ]; 1.15 (s, 3H ##STR12##

Mass spec [m/e]: 320 [M-18] , 251, 233, 221, 171, 143, 141, 137, 125,113, 97, 95, 81, 69

Chemical Ionization: M⁺ + H = 339; M.W. = 338.

(F) Preparation of I-b

The keto derivative (II-b) obtained as in C above (243 mg.) is dissolvedin tetrahydrofuran (5 ml.) and water (5 ml.). To this mixture tetran-butyl ammonium hydroxide (20% solution in methaol, 1.5 ml.) is addedunder nitrogen at room temperature and the resulting mixture is stirredfor 16 hrs. The mixture is treated with 50 ml. of ether and 20 ml. ofwater. The organic layer is separated, dried (MgSO₄) and evaporated invacuo to give an oil. This crude product is further purified bychromatography on a SilicAR column (10 g.). The product I-b (152 mg.) iseluted with ether. Its ir, nmr spectra, R_(f) on thin layer andretention time on gas chromatography are identical to those of compoundII, reported in co-pending application Ser. No. 672,918. The compoundhas the following spectral characteristics:

I.R. (Neat) μ : 2.90, 5.96 and 6.21; N.M.R. _(TMS) ^(CDCl).sbsp.3 δ:6.11 (broad s, 1H, ##STR13## 5.48 (m, 1H, >C═CH--CH₂ OH); 4.19 (d, 2H,>C═CH--CH₂ OH); 4.13 (s, 2H ##STR14## 3.56 [broad t, 1H, ##STR15## 2.10(d, 3H, ##STR16## 1.13 (s, 3H, ##STR17## 1.07 [d, 6H, ##STR18##

Mass Spec [m/e]: 334 [M-18] , 225, 240, 111, 95, 81, 69.

U.V. - A max (EtOH): ˜ 239 nm [ε=8500] .

Chemical Ionization: M⁺ + H = 353; M.W. = 352.

A. PREPARATION OF CRUDE EXTRACT

Ten kg. of dried or fresh leaves from the zoapatle plant (Mantanoatomentosa) and 30 gallons of water are added to a 100 gallonsteam-jacketed stainless steel tank. The mixture is heated at 90°-100° Cfor 2.5 hours with periodic stirring. The hot mixture is filteredthrough gauze to afford a clear dark tea, about 25 gallons in volume.The solid residue in the tank is washed with 4 gallons of hot water,filtered, and the filtrate combined with the tea obtained above. Thecombined aqueous extracts are extracted with 30 gallons of ethylacetate. The mixture is stirred vigorously and allowed to settle. Thetop frothy layer is siphoned off to break the emulsion, and as muchethyl acetate separated as possible. Another 20 gallons of ethyl acetateare added to the mixture and the above process repeated. The combinedethyl acetate extracts are evaporated at 50° C under vacuum. The residueis extracted with three portions of hot (75°-80°) benzene (10 literstotal). The benzene extracts are evaporated at 50° C under vacuum andthe residue is washed three times with a total of 8 liters of refluxinghexane. The hexane-washed residue is dissolved in 2 liters of acetone,10 g. of Nuchar is added, and the mixture is stirred for 1 hour at roomtemperature. The charcoal is removed by filtration, and the filtrateevaporated by distillation at 30° C under vacuum to afford 69 g. ofcrude extract.

B. PREPARATION OF SEMI-PURIFIED MATERIAL

The crude residue obtained in A above (50 g.) is dissolved in ether (5l.) and the resulting solution is filtered and washed with saturatedsodium bicarbonate solution (500 ml.). The ether is dried over anhydroussodium sulfate, filtered and concentrated to dryness to afford a lightyellow oil (44.6 g.). This oil is then dissolved in chloroform (400 ml.)and the solution added to a column (4 in. × 4 ft.) of 2.5 kg. of neutralsilicic acid packed in chloroform. The column is eluted with chloroform,chloroform-isopropanol mixtures, and 110 fractions are collected. Thefractions are evaporated to dryness in vacuo at a temperature below 40°C. The column is eluted as follows:

    ______________________________________                                                   Volume/                                                                       Fraction                                                           Fraction   (ml.)      Eluent                                                  ______________________________________                                        1-7        650        CHC1.sub.3                                               8-30      500        isopropanol:CHC1.sub.3 (1:41.7)                         31-60      500        isopropanol:CHC1.sub.3 (1:33.3)                          61-105    500        isopropanol:CHC1.sub.3 (1:28.6)                         106-110    500        isopropanol:CHC1.sub.3 (1:25)                           ______________________________________                                    

The composition of the fractions is monitored by thin layerchromatography [silica gel, isopropanol-chloroform (1:12.5)] and by gaschromatography -- 3% OV17 [methyl silicone - phenyl silicone (1:1)]column using a programmed run (150°-250°). Fractions Nos. 78-84 arecombined and the solvent removed in vacuo to afford an oily residue (5.1g.) which contains at least three major components as indicated by gaschromatography.

A portion of the residue (3.2 g.) is then dissolved in benzene (50 ml.)and the solution added to a column (4 in. × 35 in.) packed with 2 kg. ofOR-PVA Merck-O-Gel 2000 (A vinyl acetate copolymer which swells inorganic solvents, produced by E. M. Merck, Inc. and sold under thetrademark EM Gel® Type OR-PVA.) prepared in benzene. The column iseluted with benzene and a total of 47 fractions is collected. Thin layerchromatography and gas chromatography are used to monitor thecomposition of the fractions.

    ______________________________________                                                         Volume                                                       Fractions        Fraction (ml.)                                               ______________________________________                                        1-7              1000                                                          8-45             300                                                         46-47            1000                                                         ______________________________________                                    

Fractions 23-33 contain 1.73 g. (54%) of the applied material.

(1) Fractions 24-25 are evaporated to give compound I-b as an oil (0.251g.).

(2) Fraction 31 is evaporated to give compound I-a as an oil (0.326 g.).

The following general procedure is a standard procedure employed todetect uterine contractions in female animals.

PROCEDURE I

Mature female New Zealand rabbits are anesthetized with sodiumpentobarbital and ovariectomized. Following a recovery period of oneweek, the rabbits are treated with 8 μg./day s.c. of 17β-estradiol for 6consecutive days, followed by treatment with 1.0 μg./day s.c. ofprogesterone for 7 consecutive days. The uterus and oviducts of therabbits are perfused 72 hours after the last dose of progesteroneaccording to the method of Heilman, et al., (Fertil. Steril. 23:221-229)with slight modifications. The oviduct and uterus are perfused at a rateof 53 μl./min. The uterus is perfused with a tube extending 1.0 cm. intothe lumen of the uterus from the oviducal end. The uterus is ligated atthe utero-tubal junction. Another cannula is inserted 1.0 cm. into theuterus through a small incision in the vagina in order to collectperfusate. The material to be tested is administered i.v. through thejugular vein in a vehicle that contains polyethylene glycol 200,polyethylene glycol 400, ethanol and a phosphate buffer. The cannula isattached to a P23-Dc Stathan transducer which in turn is coupled to aGrass Model 5 polygraph and the uterine contractility measured.

Intravenous administration of the compound obtained from Fraction 31(I-a) is effective in inducing uterine contractions and relaxing theoviduct in 72-hour progesterone withdrawn rabbits in a dose range of1.0-4.0 mg./kg. The compound obtained from Fractions 24-45 (I-b) iseffective when administered in a dose range of from 25-40 mg./kg.

The following general procedure is a standard procedure employed todetect interruption of pregnancy after implantation has occurred.

PROCEDURE II

Mature, Hartley strain, female guinea pigs are continuously cohabited(monogamously) with males until a vaginal plug (copulation plug) isfound in the cage. This time is considered to be day 1 of gestation.Groups of 5-6 females are given test materials intraperitoneally in thevehicle described in Procedure I on day 22 of gestation. The pigs aresacrificed between the 25th and 45th day of gestation and examined forevidence of resorption or abortion.

Intraperitoneal administration of the material obtained from Fraction 31(I-a) is effective in interrupting pregnancy when administered in a doserange from 25-85 mg./kg.

What is claimed is:
 1. The method of purifying residues containingbiologically-active materials obtained by extraction of the zoapatleplant which comprises the steps of:treating the residue with anacylating agent selected from the group consisting of aliphatic acylhalides and aliphatic acid anhydrides having 1-10 carbon atoms in theacyl group in the presence of a base selected from the group consistingof trialkylamines, pyridine, N,N-dimethylanaline and N,N-diethylanaline,reacting the resultant mixture first with a reducing agent selected fromthe group consisting of sodium borohydride and lithium tritertiarybutoxyaluminum hydride and then with manganese dioxide and separating thecomponents of the mixture to yield a first compound having two acylgroups and a keto group and a second compound having two acyl groups anda hydroxyl group.
 2. The process of claim 1 wherein the acylating agentis an acyl halide.
 3. The process of claim 2 wherein the acylating agentis acetyl chloride.
 4. The process of claim 1 wherein the acylatingagent is an acid anhydride.
 5. The process of claim 4 wherein theacylating agent is acetic anhydride.
 6. The process of claim 1 whereinthe base is pyridine.
 7. The process of claim 1 wherein the reducingagent is sodium borohydride.
 8. The process of claim 1 whichadditionally comprises treating the compound having two acyl groups anda hydroxyl group with an oxidizing agent selected from the groupconsisting of chromium trioxide-sulfuric acid and chromiumtrioxide-pyridine and hydrolyzing the resultant ketone with a baseselected from the group consisting of sodium hydroxide, potassiumhydroxide and tetra n-butyl ammonium hydroxide to form the dihydroxycompound.
 9. The process of claim 8 wherein the oxidizing agent ischromium trioxide-sulfuric acid.
 10. The process of claim 8 wherein thebase is tetra n-butyl ammonium hydroxide.
 11. The process of claim 1which additionally comprises hydrolyzing the compound having two acylgroups and a keto group with a base selected from the group consistingof potassium hydroxide, sodium hydroxide and tetra n-butyl ammoniumhydroxide to form dihydroxy compound.
 12. The process of claim 11wherein the base is tetra n-butyl ammonium hydroxide.
 13. The method ofpurifying residues containing biologically-active materials obtained byextraction of the zoapatle plant which comprises the steps of:treatingthe residue with an acylating agent selected from the group consistingof aliphatic acyl halides and aliphatic acid anhydrides having 1-10carbon atoms in the acyl group in the presence of a base selected fromthe group consisting of trialkylamines, pyridine, N,N-dimethylanalineand N,N-diethylalanine, reacting the resultant mixture first with areducing agent selected from the group consisting of sodium borohydrideand lithium tritertiarybutoxy aluminum hydride and then with manganesedioxide, chromatography the reaction mixture over a column of adsorbentmaterail selected from the group consisting of silica gel, alumina andflorisil and collecting the fractions containing the biologically-activematerials to yield a first compound having two acyl groups and a ketogroup and a second compound having two acyl groups and a hydroxyl group.14. The process of claim 13 wherein the adsorbent material is silicagel.
 15. The process of claim 13 wherein the acylating agent is acetylchloride and the reducing agent is sodium borohydride.
 16. The processof claim 13 wherein the base is pyridine.
 17. The process of claim 13which additionally comprises hydrolyzing the compound having two acylgroups and a keto group with a base selected from the group consistingof potassium hydroxide, sodium hydoxide and tetra n-butyl ammoniumhydroxide to form the dihydroxy compound.
 18. The process of claim 17wherein the base is tetra n-butyl ammonium hydroxide.
 19. The process ofclaim 13 which additionally comprises treating the compound having twoacyl groups and a hydroxyl group with an oxidizing agent selected fromthe group consisting of chromium trioxide-sulfuric acid and chromiumtrioxide-pyridine and hydrolyzing the resultant mixture with a baseselected from the group consisting of potassium hydroxide and tetran-butyl ammonium hydroxide to form the dihydroxy compound.
 20. Theprocess of claim 19 wherein the base is tetra n-butyl ammoniumhydroxide.
 21. The process of claim 19 wherein the oxidizing agent ischromium trioxide-sulfuric acid.